Mu is a temperate bacteriophage which infects Escherichia coli and several other Gram negative enteric bacteria. It is an extraordinary phage in several respects and has carved a special niche for itself both as a genetic tool and a paradigm in phage biology, almost rivaling phage lambda. It is also a predator that has adapted its hunting skills well in order to have an extraordinarily wide host range. While phage Mu finds a mention in almost every genetics textbook for several of its unique and well-studied characteristics, there are a few aspects of its biology that are far from understood. In this thesis, light has been shed on one such less understood feature of Mu biology, namely its anti-restriction function. The enigmatic mom gene of bacteriophage Mu is the center of this thesis work. Bacteriophages, through their sheer number and versatility of attack tactics, constitute an overwhelming threat to bacteria in the natural environment. While it is not always possible to completely prevent the entry of foreign DNA into the cell, it is in the interest of the bacterium to tame the xenogeneic DNA, whose expression may have adverse effects on bacterial fitness. Bacterial nucleoid associated proteins (NAPs) participate in chromosome structuring and global transcriptional regulation. Besides this canonical role, they furnish the job of regulating xenogeneic DNA as well. NAPs are known to regulate the expression of toxin-antitoxin modules, pathogenicity islands and other horizontally transferred DNA and have a profound role in regulating transposon dynamics and the lifestyle of many phages. Chapter 1 introduces the role of bacterial NAPs in silencing foreign DNA, especially after the DNA establishes itself in the host. This thesis examines the role of a bacterial NAP namely Fis in fine-tuning an immune evasion function of bacteriophage Mu. A general introduction to phage Mu and its host expansion strategies, with special focus on its DNA modification function is also presented. Owing to the various immune evasion strategies, phages often have an upper hand on their hosts in the ongoing evolutionary arms race. One such strategy is DNA modification which bacteriophages have evolved as a means to protect their genomes from restriction enzymes of the host. While most phages employ the commonplace methylation modification for their anti-restriction function, phage Mu employs an unusual acetamido modification, catalysed by its protein Mom. Mom modified DNA is refractory to several restriction enzymes from different bacterial species. However, the modification is toxic to the host and thus mom expression needs to be precisely regulated to prevent untimely expression. A crowded multifactorial regulatory circuit has evolved to ensure the expression of mom without jeopardizing the welfare of the bacterial host. Chapter 2 uncovers a new player in mom regulation. The study shows that the bacterial chromatin architectural protein Fis is a transcriptional repressor of mom promoter and that Fis mediates its repressive effect by denying access to RNA polymerase at mom promoter. Two distinct roles of Fis have been known previously in Mu biology. In addition to bringing about the overall downregulation of transposition events and transcription of early genes of phage Mu, Fis also stimulates tail fiber flipping by aiding the activity of a site-specific recombinase. The present study thus presents a novel facet of Fis function in Mu biology. While the regulation of mom has been a matter of intense investigation over the past few decades, most biochemical and structural aspects of the Mom protein per se have remained mysterious owing to the difficulties in cloning this toxic gene. Chapter 3 describes the expression, purification and biophysical characterization of Mom. A variety of techniques show Mom to be folded and dimeric in solution. SPR studies with Mom indicate its high affinity binding to DNA. Chapter 4 deals with the attempts to identify the elusive co-factor of Mom. To begin with, the in vivo activity of Mom was demonstrated by employing a simple plasmid cleavage assay based on the resistance of Mom modified DNA to certain restriction endonucleases. A variety of disparate in silico structure prediction tools such as I-TASSER, Robetta and PHYRE indicate Mom to be related to the GCN5-related N-acetyltransferase superfamily. Mutation of residues deemed important from this analysis indeed abolished or reduced Mom activity in vivo, validating the bioinformatics based prediction and shed light on the possible active site of Mom. However, acetyltransferases are not known to transfer acetamido groups. It was also necessary to establish beyond doubt, the chemical structure of the Mom modified nucleoside. High resolution mass spectrometry data showed the modification to be acetamido, corroborating the earlier sole report on this aspect. Based on the biochemical reactions that acetyl coenzyme A is known to participate in, it is difficult to explain the involvement of acetyl coenzyme A in acetamido addition. Notwithstanding the converging predictions of different bioinformatics tools, caution is recommended when inferring function from structurally similar family members. It is possible that a different chemistry might have converged on the same (acetyltransferase) fold, given that none of the known pathways utilizing acetyl coenzyme A can explain the Mom modification. Several likely candidates such as carboxy-SAM, glyoxylic acid and glycine were also tested for being donors of the two carbon entity transferred on adenine by Mom. Since these candidates tested negative in our genetic assays, a genome-wide genetic screen was subsequently devised to identify the host genes involved in mom modification. The assay exploited the phenotype of lethality associated with overexpression of Mom in E. coli in order to screen for mutations in the host genome that rescued the toxicity. However, the survivors which were obtained in this assay had emerged through mutations in the mom gene rather than abrogation of the co-factor synthesis pathway of the host. The results point at two possibilities: (i) utilization of essential gene(s) or (ii) existence of redundant pathways for the Mom modification reaction. Chapter 5 is an account of our attempts to trace the lineage of mom and its regulatory region, employing updated DNA and protein sequence databases. Despite the selective advantage conferred on the phage by the anti-restriction function of mom, in many Mu-like phages, mom is either absent or substituted with methyltransferases. However, in Mu-like genomes that do encode mom, in spite of a significant overall sequence divergence from Mu, the core elements of the mom regulatory circuit seem to have either co-evolved or have been selectively conserved. Although Mu appears to be unique in the possession of a regulatory circuit tailored for the purpose of mom regulation, recently discovered Mu-like genomes show that different types of regulatory features evolved several times in closely related genomes. It is very likely that a toxic gene like mom has earned its place in the phage genome by carrying along with itself a baggage of regulatory elements. Failure to sustain sufficient regulatory pressure may trigger the loss or replacement of the advantageous but potentially lethal mom function.

Regulation of transcription initiation is the major determining event employed by the cell to control gene expression and subsequent cellular processes. The weak promoters, with low basal transcription activities, are activated by activators. Bacteriophage Mu mom gene, which encodes a unique DNA modification function, is detrimental to cell when expressed early or in large quantities. Mu has designed a complex, well-controlled and orchestrated regulatory network for mom expression to ensure its synthesis only in late lytic cycle. The phage encoded transcription activator protein C activates the gene by promoter unwinding of the DNA and thereby recruiting of RNAP to the promoter. C protein functions as a dimer for DNA binding and transcription activation. Mutagenesis and chemical crosslinking studies revealed that the leucine zipper motif, and not the coiled coil motif in the N terminal region, is responsible for C dimerization. The DNA binding domain of C is a HTH domain which is preceded by the leucine zipper motif. The C protein is one of the few examples in the bacterial proteins containing both leucine zipper and HTH domain. Most of the transcription activators either influence initial binding of RNAP or conversion of closed to open complex formation. Very few activators act at subsequent steps of promoter-polymerase interaction. Earlier studies showed high level of transcription from a mutant mom promoter, tin7. Addition of C further increased transcription from Ptin7 indicating that C may have a role beyond polymerase recruitment. Each steps of transcription initiation have been dissected using the Ptin7 and a positive control (pc) mutant of C, R105D. The results revealed multi-step transcription activation mechanism for C protein at Pmom. C recruits RNAP at Pmom and subsequently increases the productive RNAP-promoter complex and enhances promoter clearance. To further understand the C mediated transactivation mechanism, interaction between C and RNAP was assessed. C interacts with holo and core RNAP only in presence of DNA. Positive control mutants of C, F95A and R015D, were found to be compromised in RNAP interactions. These mutants were efficient in RNAP recruitment to Pmom but do not enhance promoter clearance. Trypsin cleavage protection experiment indicated that probably C protein interacts with b¢ subunit of RNAP. Interaction between C and RNAP appears to enhance the formation of productive RNAP-promoter complex leading to promoter clearance. The connection between activator-polymerase interaction and transcription activation is well documented where the recruitment of RNAP is influenced. In case of activators acting at post recruitment steps of initiation, the role of polymerase contact is poorly understood. Our study shows that activator-polymerase interaction can lead to increased promoter clearance at Pmom by overcoming abortive initiation.